Two enzymes each having an activity of releasing D-glucose linked at a non-reducing end through an α-1,4 bond, glucan 1,4-α-glucosidase and α-glucosidase, are known.
Glucan 1,4-α-glucosidase (EC 3.2.1.3) is also called 1,4-α-D-glucan glucohydrolase or glucoamylase. It is an enzyme-that acts on a non-reducing end of a polymer of D-glucopyranose linked through α-1,4 bonds to release β-D-glucose. Glucan 1,4-α-glucosidases derived from the following are currently known: fungi such as those of genus Aspergillus, genus Mucor, genus Rhizopus, genus Piricularia, genus Thermomyces and genus Trichoderma; yeasts such as those of genus Endomyces and genus Saccharomyces; and a bacterium of genus Clostridium. This enzyme, like α-amylase, is an important enzyme used in a process of hydrolyzing starch. Thus, the enzyme is industrially utilized in wide variety of fields including production of glucose, isomerized sugars and oligosaccharides, as well as production of liquors and fermented alcohol.
Glucose is usually produced from starch as follows. Starch is gelatinized by cooking. The gelatinized starch is liquefied by allowing α-amylase to act on it at about 80 C. Then, saccharification is carried out by allowing glucan 1,4-α-glucosidase to act at 55 to 60 C. The liquefaction is carried out at a high temperature because the gelatinized starch is highly viscous and because thermostable α-amylases have been put to practical use. It is desirable to select a temperature of 55 C. or above for the saccharification in order to avoid contamination with microorganisms. However, if a commonly used enzyme derived from a fungus is used, one must select a temperature of 60 C. or below because of the thermostability of the enzyme. Accordingly, it is impossible to carry out the steps of liquefaction and saccharification at the same time because their optimal temperatures are different from each other, resulting in a great wasteful cost for energy.
α-Glucosidase (EC 3.2.1.20) is an enzyme that acts on an α-glucoside bond at a non-reducing end to release α-D-glucose. It is widely present in animals, plants and microorganisms. α-Glucosidases are classified into groups (1) to (3) based on the substrate specificity as follows: (1) ones that act on a wide variety of hetero and homo α-glucoside compounds; (2) ones that are highly specific for α-1,4-glucooligosaccharides and that have relatively weak activities on high molecular weight glucan and heteroglucoside; and (3) ones that are highly specific for α-1,4-glucoside bonds or that also act on starch or glycogen. Among these, those belonging to the group (3) are often called glucoamylases (“Oyo Kosogaku”, Tsujisaka et al. (eds), Kodansha (1979) pp. 56).
Hyperthermophilic microorganisms, which are adapted to high temperature environment, produce highly thermostable enzymes. Pyrococcus furiosus, a hyperthermophilic archaebacterium, is known to produce saccharide-hydrolyzing enzymes such as α-amylase, α-glucosidase, β-glycosidase and β-glucanase. Genes for some of these enzymes have been cloned (The Journal of Biological Chemistry, 268:24402-24407 (1993); The Journal of Biological Chemistry, 272:16335-16342 (1997); Journal of Bacteriology, 172:3654-3660 (1990); Journal of Bacteriology, 177:7105-7111 (1995); Journal of Bacteriology, 181:284-290 (1999)). However, there is no known hyperthermophilic microorganism producing glucan 1,4-α-glucosidase or hyperthermostable glucan 1,4-α-glucosidase. Furthermore, known α-glucosidases produced by hyperthermophilic microorganisms including Pyrococcus furiosus act only on low molecular weight substrates and do not digest high molecular weight substrates such as starch.